HUMAN GENE THERAPY 17:577–588 (June 2006)© Mary Ann Liebert, Inc.

Review

Gene-Based Immunotherapy for Human ImmunodeficiencyVirus Infection and Acquired Immunodeficiency Syndrome

BORO DROPULIC1 and CARL H. JUNE2

ABSTRACT

More than 40 million people are infected with human immunodeficiency virus (HIV), and a successful vac-cine is at least a decade away. Although highly active antiretroviral therapy prolongs life, the maintenance ofviral latency requires life-long treatment and results in cumulative toxicities and viral escape mutants. Genetherapy offers the promise to cure or prevent progressive HIV infection by interfering with HIV replicationand CD4� cell decline long term in the absence of chronic chemotherapy, and approximately 2 million HIV-infected individuals live in settings where there is sufficient infrastructure to support its application with cur-rent technology. Although the development of HIV/AIDS gene therapy has been slow, progress in a numberof areas is evident, so that studies to date have significantly advanced the field of gene-based immunother-apy. Advances have helped to define a series of ongoing and planned trials that may shed light on potentialmechanisms for the successful clinical gene therapy of HIV.

577

INTRODUCTION

THE RATIONALE for continued investigation into novel treat-ment and prevention strategies for human immunodefi-

ciency virus (HIV)/acquired immunodeficiency syndrome(AIDS) remains strong. Although the introduction of highly ac-tive antiretroviral therapy (HAART) has provided an effectivemeans to suppress viral replication, HAART is not curative be-cause the virus hides in latent reservoirs, ready to reinitiate theinfection even after prolonged therapy (Siliciano et al., 2003).Emergence of drug-resistant or multidrug-resistant strains ofHIV has been on the rise, as exemplified by increases in trans-mission of these viruses (Wensing and Boucher, 2003; Wens-ing et al., 2005). These caveats with HAART support the ra-tionale to develop novel approaches to treat the disease,including gene therapy.

AIDS is a disorder of the immune system that is caused bycollapse of immunity driven primarily by depletion of CD4� Tcells. Therefore, protection of the T cell compartment via ge-netic modification of T cells or stem cells is an attractive hy-pothesis for prevention of AIDS onset. This has been examined

since the 1990s in a series of phase I and phase II clinical tri-als, and continues to be enthusiastically investigated. Despitethe work that has been done, it remains theoretical as to whatthe in vivo mechanism of a successful HIV gene therapeuticwould be. Although the antiviral efficacy of genetic payloadscan easily be tested, the complex dynamics between HIV repli-cation, T cell homeostasis, and anti-HIV immunity in the bodymake our understanding of an effective clinical approach chal-lenging at best. This review is intended to provide an overviewof the payloads, vectors, and cellular targets that have been in-vestigated to date in clinical trials. We summarize lessons thathave been learned and describe where this field of research isgoing, and we illustrate some of the challenges and opportuni-ties for development of a successful treatment for HIV/AIDS.

ANTI-HIV PAYLOADS

Several classes of anti-HIV genetic payloads have beenshown to be effective in inhibition of HIV replication in vitro.These include dominant negative mutant proteins, intracellular

1Lentigen, Baltimore, MD 21227.2Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104.

antibodies, antisense, RNA decoys, ribozymes, interferingRNA, and HIV-activated T cell receptors, among some addi-tional novel approaches. There are ample choices for anti-HIVpayloads, as is shown below. When choosing an approach it isimportant to balance efficacy with durability regarding rapidvirus evolution, low immunogenicity (because an immune re-sponse would lead to rejection of genetically modified cells),and targeting of conserved regions of HIV whenever possibleto allow for panclade efficacy.

Dominant negative proteins

Dominant negative mutant proteins interfere with the HIVlife cycle. Several have been tested that target both the virusand cellular proteins that are involved in the life cycle of thevirus. The most extensively studied dominant negative mutantprotein is Rev M10, which interferes with efficient export ofunspliced genomic HIV RNA from the nucleus into progenyviral particles. Rev M10 has a mutation in a highly conserveddomain known to interact with cellular factors, and has beenshown to efficiently inhibit HIV replication in vitro (Malim etal., 1989, 1992). Cells expressing this protein have shown asurvival advantage over non-Rev M10-expressing cells in clin-ical trials in which HIV-infected individuals were infused withcells genetically modified to express the mutant protein (Rangaet al., 1998; Morgan et al., 2005).

Other viral targets include the Gag protein and other proteinsexpressed during the late stages of the HIV life cycle. A trans-dominant mutant Gag has been shown to effectively inhibit HIVin vitro (Trono et al., 1989; Cara et al., 1998). Another targetis the Vif protein, which is essential for the production of in-fectious HIV particles in lymphocytes and macrophages. Its roleis to overcome the deaminating effects of APOBEC3G proteinon the viral genome, thereby facilitating efficient infection ofcells by progeny virions. Trans-dominant mutant peptides ofVif have been developed that disrupt Vif function by interfer-ing with its oligomerization (Yang et al., 2001, 2003). Exper-iments with these mutant Vif peptides have shown significantinhibition of HIV replication, demonstrating their potential util-ity. Finally, peptides derived from HIV reverse transcriptasehave been shown to inhibit the ability of the virus to integrate,thus providing an early block to HIV replication in the cell (OzGleenberg et al., 2005).

Other studies have attempted to develop trans-dominant mu-tant proteins that interfere with cellular targets. Attractive cel-lular targets are the CCR5 receptor, which is a coreceptor forinfection of CD4� cells with CCR5 (R5) strains of HIV, andrestriction factors such as the tripartite motif protein TRIM5�(Stremlau et al., 2004). Trans-dominant mutant variants ofCCR5 have been shown to interfere with HIV infection ofCD4� T cells (Luis Abad et al., 2003). Further support for thisapproach comes from the fact that individuals homozygous fora 32-bp deletion mutant of the CCR5 receptor (�32-CCR5) aremore resistant to R5 strains of HIV than are individuals whoexpress the wild-type receptor. Another cellular target is theprotein that interacts with HIV integrase (IN), INI1. A fragmentof this protein acts in a dominant negative fashion to inhibit in-teraction of IN with the functional cellular protein (Yung et al.,2001). Finally, peripheral blood lymphocytes transduced witha simian virus 40 (SV40) vector expressing the native �1-an-

titrypsin gene were shown to decrease the processing of p55gag

and gp160env proteins, significantly decreasing HIV replication(Cordelier et al., 2003b).

Intrabodies

Intracellularly expressed recombinant antibodies, termed in-trabodies, have gained increased popularity. Intrabodies thatbind to both viral and cellular targets have been developed. Anintrabody targeted to HIV p17gag showed strong inhibition ofseveral strains of HIV (Deepanker Tewari, 2003). Several sin-gle-chain antibodies targeted to the reverse transcriptase mole-cule of HIV have been identified (Herschhorn et al., 2003), butmore studies are required to confirm their general utility forHIV gene therapy. More analysis has been performed for ananti-Vif intrabody that significantly affects its function, pre-sumably by interfering with oligomerization. Peripheral bloodlymphocytes transduced with a lentiviral vector expressing ananti-Vif intrabody was shown to significantly inhibit both lab-oratory and primary strains of HIV (Goncalves et al., 2002).

Cellular targets for intrabody gene therapy may be more at-tractive than viral targets because of virus escape mutations.Single-chain antibodies targeted to CXCR4 and cyclin T1 havebeen developed and were shown to inhibit the replication ofvarious HIV strains (BouHamdan et al., 2001; Bai et al., 2003).However, further testing is required to show that targeting suchcellular factors in the primary lymphoid cells will not result intoxicity. Intrabodies to the CCR5 receptor are also in develop-ment (Cordelier et al., 2004). A significant challenge for allprotein-based therapeutics is to avoid immunogenicity in orderto circumvent immune-mediated rejection of vector-transducedcells.

Anti-HIV RNAs

Numerous RNA-based strategies against HIV have beenevaluated. These include ribozymes, RNA decoys, antisense,and, most recently, RNA interference (RNAi). There are dis-tinct advantages to an RNA approach: (1) heterologous RNAmolecules expressed in cells are not immunogenic; (2) there areseveral types of RNA inhibitory molecule to choose from in-cluding antisense, ribozymes, decoys, and, most recently,RNAi; (3) multiple sequences on the HIV genome can be tar-geted simultaneously, addressing the issue of HIV resistance;and (4) both viral and cellular sequences can be targeted si-multaneously, offering the possibility of attacking HIV repli-cation at multiple steps of the life cycle and further against cel-lular targets that are not prone to HIV resistance.

Numerous antisense targets have been tested, targeting bothcoding and noncoding regions of the HIV genome, and mosthave been shown to effectively inhibit HIV replication (Good-child et al., 1988; Sczakiel et al., 1992; Chuah et al., 1994; Sunet al., 1995; Vandendriessche et al., 1995; Veres et al., 1996,1998; Humeau et al., 2004; Lu et al., 2004b). The size of an-tisense plays an important role in antiviral efficacy, with shortersequences showing lesser or no inhibition, and sequencesgreater than 1000 bases having the best efficacy (Goodchild etal., 1988; Veres et al., 1996). An advantage of using long an-tisense is that it is difficult for HIV to develop resistance againstit, because such extensive mutation would render the virus repli-cation incompetent or severely debilitated. This concept was

DROPULIC AND JUNE578

demonstrated in the laboratory by Lu and colleagues (2004b),where a lentiviral vector expressing long antisense targeted tothe envelope region of HIV was evaluated in an in vitro sys-tem of serial selection in order to isolate HIV resistance mu-tants. Although several mutants were isolated, none had devel-oped resistance to the antisense and only one mutant retainedthe ability to replicate autonomously. Ultimately, follow-upstudies in the clinic will be needed to fully establish the lackof HIV antisense resistance mutations.

Ribozymes are similar to antisense but have the added fea-ture of cleaving their target catalytically. Several studies haveshow inhibition of HIV replication, using ribozymes (Rossi,1999; Morris and Rossi, 2004). More recent studies have shownthat many types of ribozymes require modified structures fortrue catalytic activity, suggesting that the anti-HIV effects ofearlier studies in some cases may be the result of the innate an-tisense effects, rather than their purported catalytic properties(Khvorova et al., 2003). A ribozyme approach is likely to re-sult in HIV escape mutants if it is not used in a multipayloadsetting, or against a cellular target.

In contrast to antisense and ribozymes, decoys do not attacktarget RNAs directly; rather, they mimic RNA structures involvedin the viral life cycle and decoy viral or cellular factors awayfrom propagation of the virus. HIV has several cis-acting nucleicacid sequences, and two in particular specifically interact withthe HIV proteins Tat and Rev and have been tested as antivirals(Sullenger et al., 1990, 1991; Lee et al., 1994; Bahner et al.,1996; Bukovsky et al., 1999; Li et al., 2005). HIV Tat binds tothe trans-acting response element (TAR) sequence to facilitatetranscriptional elongation for production of full-length HIVgenomes. Rev binds to the Rev response element (RRE) to fa-cilitate the export of unspliced genomic RNA from the nucleus.Both of these interactions are critically involved in the replica-tion cycle of the virus. The RRE has been stably expressed incells via murine leukemia virus (MLV) retroviral vectors andshown to interfere with HIV replication (Bahner et al., 1996).TAR decoys expressed by lentiviral vectors are presently antic-ipated for clinical testing in a triple payload vector construct (Liet al., 2005). Of note, HIV-based lentiviral vectors are generallyconfigured to contain TAR and RRE elements, so even withoutadditional anti-HIV sequences they express cis-acting elementsthat can decoy factors away from replication of infectious HIV.

More recently, RNAi has become a powerful tool for genesilencing, and the first report regarding its utility against HIVwas made in 2002 (Das et al., 2004). Since that time, improvedmethods for expression of RNAi by gene therapy vectors, mostnotably short hairpin RNAs (shRNAs) driven by Pol III pro-moters such as the U6 promoter, have enabled the practical ap-plication of this technology (An et al., 2003). As with antisenseand ribozymes, RNAi has been used to degrade various viraland host cell targets involved in the life cycle of HIV-1 (Coburnand Cullen, 2002; Novina et al., 2002). However, targeting HIVRNA with shRNA, although effective in the short term, is prob-lematic for long-term inhibition of HIV replication as even asingle point mutation on the target RNA can dramatically af-fect the efficacy of the shRNA molecule, and by now many in-vestigators have reported RNAi escape (Boden et al., 2003; Daset al., 2004). Given the diversity of HIV strains and the easewith which HIV mutates, single-modal shRNAs that target HIVare not viable for use in HIV gene therapy.

To address this issue, Rossi and colleagues developed mul-titargeted RNA interference-based lentiviral vectors that targetboth HIV and cellular proteins involved in the life cycle of HIV(Li et al., 2005). In this study, the combination of a Pol III U6promoter-driven shRNA targeting the rev and tat mRNAs ofHIV-1, a U6-transcribed nucleolar-localizing TAR RNA decoy,and a VA1-derived Pol III cassette that expresses an anti-CCR5ribozyme was engineered into a self-inactivating lentiviral vec-tor. The study demonstrated that the combination vector waseffective in controlling the replication of HIV and appeared toprevent the production of escape mutants. A clinical trial to testthis approach is in preparation (www4.od.nih.gov/oba/rac/min-utes/RAC_minutes_09-05.pdf).

Other approaches

A potential mechanism for success in HIV gene therapy in-volves the boosting of HIV immunity to enable the body to con-trol virus replication and spread. Typically this is attempted viatherapeutic vaccination, but these approaches have historicallybeen unsuccessful. Studies initiated in the early 1990s exam-ined the potential of engineering HIV-specific cytotoxic T lym-phocytes (CTLs), using the CD4 extracellular domain or a gp41-specific antibody coupled to the � signaling chain of the CD3T cell receptor (Roberts et al., 1994; Yang et al., 1997). Thesepreclinical studies demonstrated the proof of principle that theredirected CD8� T cells were capable of responding by inter-leukin (IL)-2 secretion on binding to HIV, and exhibited robustHIV CTL activity that equaled or superseded historical CTLcontrols. The CD4–CD3� approach was then translated to clin-ical application (Mitsuyasu et al., 2000; Walker et al., 2000;Deeks et al., 2002).

It has been proposed that blocking of an early step in theHIV life cycle will be important to confer a selective advan-tage to vector-modified cells in the body, and hence allow out-growth of HIV-resistant cells in the patient (von Laer et al.,2006). There are few antiviral genes available that block HIVpreintegration. The new antiretroviral drug enfuvirtide, com-monly known as T20, has been released to the market (Lalezariet al., 2003). T20 blocks HIV entry by inhibiting the confor-mational changes needed for fusion of the viral envelope withthe cellular membrane (Eckert and Kim, 2001). For a geneticapproach, the T20 peptide was modified with an anchor pro-tein for cell surface expression, and further optimized for re-duced immunogenicity and improved expression and stability,and this optimized peptide was called M87o (Egelhofer et al.,2004). An issue is that, although resistance has not yet beendocumented in the gene therapy setting, the fact that resistanceis characterized after T20 treatment generates some concernover the approach if it is used without a multitargeted payload(Poveda et al., 2004; Sista et al., 2004). Therefore it may beimportant to use anti-HIV surface peptides in combination withother surface inhibitors or other modalities to interfere with thevirus replication cycle.

There are several additional modalities that have shown pre-clinical promise, using unconventional approaches for haltingHIV replication or spread. One interesting approach places theherpes simples type 1 thymidine kinase (TK) gene under thecontrol of the HIV long terminal repeat (LTR). After HIV in-fection and in the presence of acyclovir, the HIV-infected cells

GENE-BASED IMMUNOTHERAPY FOR HIV/AIDS 579

died before the virus could spread in the culture, offering aunique approach for deletion of HIV-containing cells in the pa-tient (Caruso and Klatzmann, 1992). A second approach alsoused the HIV LTR to trigger conditional gene expression of in-terferon-�, which interferes with the subsequent round of in-fection and hence spread of HIV (Sanhadji et al., 1997; Corde-lier et al., 2003a). Another group designed an HIV LTR-specifictranslational inhibitor (Segal et al., 2004).

Studies have shown that the expression of simian TRIM5�,which binds to the HIV capsid and interferes with the uncoat-ing process, strongly protects human cells from productiveHIV-1 infection (Stremlau et al., 2004). The human version ofTRIM5� is not efficient at blocking HIV, presumably becausethe capsid protein has evolved to reduce the interaction. Thisis also observed with decreased binding of the simian immu-nodeficiency virus (SIV) capsid to the simian TRIM5� whencompared with HIV capsid binding to the simian protein. Be-cause the simian protein has a high level of homology to thehuman protein, it is anticipated that gene therapy using this genemay not be immunogenic.

Worthy of consideration in light of heightened awareness ofrisks associated with integrating vectors, zinc finger-bindingproteins fused to a nuclease domain (zinc finger nucleases) havebeen developed and proof of principle was established for thetreatment of immunodeficiency due to common �-chain genemutations (Urnov et al., 2005). This is a “hit and run” approachbecause only genetic deletion occurs, thus obviating safety con-cerns about potential adverse effects of vector integration. Zincfinger nucleases targeting the CCR5 locus are now being eval-uated in preclinical development for ultimate clinical applica-tion for patients with HAART-resistant strains of R5-tropicHIV-1.

CLINICAL VECTORS FOR HIV GENE THERAPY

Similar to HAART, with the exception of gene therapy-basedvaccines, any successful gene therapy for HIV requires dura-bility because of the latent stage of the virus. Accordingly, onlyintegrating viral vectors have been used to date in gene ther-apy trials for HIV/AIDS: murine leukemia virus (MLV)-basedretroviral and HIV-based lentiviral vectors. Although both vec-tor types are derived from the Retroviridae family of viruses, aconvention has evolved to call vectors derived from MLV“retroviral vectors” whereas vectors derived from lentivirus arecalled “lentiviral vectors.” Until recently, all the HIV gene ther-apy trials have employed MLV vectors (Table 1).

HIV clinical trials that have employed MLV vectors estab-lished that the vectors are well tolerated and reported no vec-tor-related adverse events. Although these vectors have beensuitable, a disadvantage has been that quiescent cells cannot bereadily transduced because the MLV preintegration complexrequires cell division for access to the cellular genome for in-tegration (Roe et al., 1993). Also, MLV vectors integrate pref-erentially near transcriptional start sites (Mitchell et al., 2004),increasing the possibility for insertional mutagenesis throughactivation of putative oncogenes.

We predict that lentiviral vectors will be the preferred choicein the future for reasons of safety and efficacy. Lentiviral vec-

tors not only can transduce nondividing cells, but also can trans-duce stimulated cells with efficiencies exceeding 90%, abro-gating the need for selection of transduced cells and improvingthe potential effectiveness of each cellular dose (Humeau et al.,2004). Like retroviral vectors, lentiviral vectors can be pseudo-typed with envelope proteins that broaden their tropism and sta-bilize the vector during its manufacture (Yee et al., 1994; Burnset al., 1993).

Lentiviral vectors can be derived from the feline immunode-ficiency virus (FIV), equine infectious anemia virus (EIAV), bo-vine immunodeficiency virus (BIV), simian immunodeficiencyvirus (SIV), and the human immunodeficiency virus. Only HIV-derived lentiviral vectors have been used in a clinical setting, al-though lentiviral vectors based on non-HIV species have beendeveloped (e.g., SIV, BIV, EIAV, and FIV). One perceived ad-vantage of non-HIV-based lentiviral vectors is that they are notknown to cause disease in humans, and therefore could be safer.However, because the consequence of a recombination betweenan animal lentiviral vector and wild-type HIV in the event of in-fection of transduced cells is unknown, there remains concernthat this could lead to the creation of new human pathogens. Atleast in the case of HIV, recombination between the same typesof viruses has occurred and the outcome is well understood.

In addition to improved efficacy of gene transfer, it is an-ticipated that lentiviral vectors have an improved safety pro-file in terms of insertional oncogenesis. Preclinical data haveshown that lentiviral vectors do not have the enhancer activ-ity in their promoter region that retroviral vectors have, whichcould contribute to distal aberrant gene expression in the cell(Lu et al., 2004a). In addition, the insertion profile of lentivi-ral vectors tends to occur more within the gene coding re-gion, where gene disruption instead of activation would hap-pen, instead of upstream in the 5� noncoding region, such asretroviruses are known to do (Mitchell et al., 2004). Finally,MLV has been historically known to have oncogenic poten-tial that is related to its insertion (Tsichlis, 1987). In starkcontrast, natural HIV infection is not associated with T cellleukemogenesis. However, more clinical data with long-termfollow-up are needed to establish the safety of lentiviral vec-tors with confidence.

Clinical experience with HIV-based lentiviral gene therapy

Because of the perceived risks described above, only in 2003was the first lentiviral vector trial initiated. This vector ex-pressed a long antisense against the HIV envelope gene in au-tologous CD4� T cells. This trial has been concluded, and itshowed that the patients tolerated the lentiviral vector-modifiedT cells well and that there were no adverse events associatedwith the vector (MacGregor et al., 2005; Manilla et al., 2005).Three of five subjects had prolonged engraftment with lentivi-rally modified T cells for at least 1 year after infusion, and al-though no statistically significant anti-HIV effects can be ob-served in a pilot trial, it is notable that one patient developed asustained decrease of �1.5 log in viral load. A second phaseI/II trial has just been initiated to evaluate the therapy in thecontext of structured treatment interruption (STI) and a follow-up phase II repeat dosing exploratory trial is in progress. Twoadditional unrelated lentivirus vector-based trials are antici-

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CELLULAR TARGETS

For HIV, all gene therapy trials have used ex vivo gene trans-fer in part because there does not yet exist efficient in vivo tar-geting for gene therapy. In addition, ex vivo gene transfer per-mits optimized gene transfer efficiency; reduces the risk of genetransfer to nontarget cells, which is a particular safety issue forintegrating vectors; and typically reduces or avoids develop-ment of immunity against the vector because it is washed outby the time the patient receives the product. Ex vivo cellulartargets for HIV gene therapy have historically been either Tlymphocytes or stem cells (Table 1). The advantage to trans-ducing stem cells is that then all blood cell progeny will ex-press the antiviral gene, thus conferring resistance to all HIV-susceptible cells including T cells, monocytes, macrophages,dendritic cells, and microglial cells. Using T cells as the genetherapy vehicle offers some advantages including the potentialto augment anti-HIV immunity, and the ability to expand thecells and increase each dose size to improve engraftment and,it is hoped, the therapeutic effect.

T cells

Significant advances have been made in the manipulationand growth of T cells ex vivo. In particular, the discovery thatthe anergy induced by stimulation of T cells with CD3 alonecould be overcome through costimulation of the CD3 and CD28receptors enabled large-scale amplification of T cells (Boise etal., 1995; Levine et al., 1995, 1997; Noel et al., 1996; Rad-vanyi et al., 1996). Furthermore, CD28 costimulation inducesa state of resistance to HIV infection in CD4� cells (Carroll etal., 1997). The feasibility of T cell processing for productionof sufficient doses of cellular product to conceivably have aclinical impact has been demonstrated (Levine et al., 1995,1996, 2002). The T cell-based HIV gene therapy trials to datehave reported no or modest effects on viral load, but have es-tablished an encouraging body of data supporting safety, a se-lective advantage of gene-modified HIV-resistant T cells in thebody, and also the ability of the cells to persist long term.

CD4� T lymphocytes were first genetically modified to ex-press the trans-dominant negative protein Rev M10 in a clini-cal trial in which autologous CD4� T cells were modified withgold microparticles or by an MLV vector expressing the mod-ified Rev M10. This trial was novel in that patient cells weretransduced with either a vector expressing the antiviral gene, ora marking vector. A mixture of both cell populations was givento the patient. This trial demonstrated a selective advantage inthe transduced cells containing the antiviral gene, but not thecontrol transduced cells, in patients chronically infected withHIV. Rev M10-expressing cells were detectable for an averageof 6 months compared with 3 weeks with control cells (Rangaet al., 1998).

In subsequent trials, the CD4–CD3� construct was used tomodify CD4� and CD8� T cells in patients via MLV vectordelivery. One phase I trial evaluated syngeneic cell transfer ofone to six infusions of gene-modified cells, and the second trial

evaluated a single larger infusion of autologous product (Mit-suyasu et al., 2000; Walker et al., 2000). This second study es-tablished lymphoid tissue trafficking and in vivo persistence ofmodified cells through the analysis of rectal mucosal biopsies.Stable engraftment was demonstrated, as the CD4–CD3� genewas detected in 1–3% of blood mononuclear cells at 8 weeksand 0.1% at 1 year postinfusion (Mitsuyasu et al., 2000). A ran-domized phase II study of the CD4–CD3� vector was then con-ducted in a total of 40 patients (20 treated and 20 control pa-tients), and it confirmed that the T cell infusions resulted inelevated CD4� T cell counts and the stable persistence of vec-tor-modified cells. Furthermore, it demonstrated a modest anti-viral effect (p � 0.07) on HIV rebound in these well-controlledpatients (Deeks et al., 2002). These trials, together with anothernongene transfer HIV cellular therapy trial not discussed here(Levine et al., 2002), helped to establish the safety of multipleinfusions of activated T cells in HIV patients.

More recently, Morgan and colleagues reported long-termengraftment with T cells engineered to express an antisenseTAR element or Rev M10 (Morgan et al., 2005). Robust anti-viral effects were documented, particularly in patients with highviral loads. Macpherson et al. have reported persistent engraft-ment of T cells for more than 4 years after treatment of syn-geneic CD4� T cells with an MLV vector expressing an anti-tat ribozyme (Macpherson et al., 2005). This study was similarto the Rev M10 study in that cells were transduced either withan empty vector or a vector expressing an anti-HIV payload. Incontrast to Ranga et al. (1998) and Morgan et al. (2005), how-ever, Macpherson et al. reported no selective advantage of theHIV-resistant cells when compared with control cells.

The most recent HIV gene therapy trial evaluated a retrovi-ral vector (M87o) that encodes the membrane-anchored antiviralpeptide C46. C46 comprises 46 amino acids and is derived fromthe second heptad repeat of the HIV-1 envelope glycoproteingp41 and inhibits fusion of the viral and cellular membranes dur-ing virus entry (Egelhofer et al., 2004). A pilot clinical trial wascarried out by von Laer, van Lunzen, and colleagues in 10 pa-tients with late-stage HIV/AIDS and HAART failure, who weregiven an infusion of CD4� T cells transduced with the retrovi-ral vector. Initial results from the study indicate that the approachis safe, and that although a significant rise in CD4� T cell countswas seen, viral loads were not affected. Gene marking could bedetected throughout the 1-year follow-up.

Stem cells

Human hematopoietic progenitor cells (HPCs) represent animportant cellular target for HIV/AIDS gene therapy. In con-trast to T cells, the genetic modification of stem cells offers thepotential to interfere with HIV replication in the multiple celltypes that are the targets of HIV infection such as monocytes,macrophages, dendritic cells, and microglial cells, all of whichare derived from HPCs. Furthermore, HPC therapy has the po-tential to improve the T cell receptor repertoire after thy-mopoiesis and the production of genetically resistant cells, aparticular advantage for patients with severe T cell depletion.However, at present use of HPCs as a cellular vehicles for HIVgene therapy is limited by several factors including low genetransfer efficiency of MLV vectors, silencing of anti-HIV se-quences, and impaired engraftment of primitive multipotential

GENE-BASED IMMUNOTHERAPY FOR HIV/AIDS 583

cells after transplantation (Case et al., 1999; Ellis, 2005). Someof these limitations can be addressed by improved vector tech-nology and encouraging results have been reported, as describedbelow.

An early study of MLV-transduced CD34� marrow cells expressing an RRE decoy resulted in the marking of0.001–0.003% of peripheral blood cells, which dropped evenlower after 6 months (Kohn et al., 1999). This study indeedhelped to establish the tolerability of this approach, but the long-term persistence of the cells was disappointing. A separate studywith MLV vectors in the setting of bone marrow transplanta-tion for hematologic malignancies in patients with HIV reportedpersistence to more than 2 years, perhaps because of the im-proved engraftment due to the myeloablative conditioning(Kang et al., 2002).

Later studies have shown more promise, even in the absenceof conditioning before HPC transplantation. CD34� cells iso-lated from mobilized peripheral blood and transduced with anMLV vector expressing a ribozyme against tat demonstratedthat HIV patients are capable of significant levels of thymicmaturation, supporting the important hypothesis that the CD4�

T cell compartment can be protected through HPC modifica-tion (Amado et al., 2004). A second study performed in twochildren used the dominant negative Rev M10 gene or a con-trol MLV vector for modification of autologous bone marrowCD34� cells. Vector-modified cells were rarely detected afterabout 3 months, but intriguing results showed the reemergenceof vector-modified cells in peripheral blood mononuclear cells(PBMCs) after an increase in HIV replication (Podsakoff et al.,2005). Only cells modified with the anti-HIV gene were de-tected, which provides in vivo evidence for HIV-induced se-lection of gene-modified cells.

CHALLENGES AND FUTURE DIRECTIONS

Many approaches have been developed for inhibition of HIVin tissue culture; however, few of these have been translatedfor clinical evaluation. After more than a decade of investiga-tion into these trials, a successful treatment for HIV gene ther-apy remains on the horizon. Contrary to what some may con-sider slow progress in HIV gene therapy, the field has advancedin a meaningful way through the observations made in past tri-als regarding cellular persistence as it relates to the cellular ve-hicle, the dose and antiviral payload, and the safety of cellulartherapy with retroviral and lentiviral vectors.

Although there have been some encouraging results, the ma-jor difficulty remains in understanding the mechanism thatmight lead to success in the clinic. Without understanding sucha mechanism, it remains difficult to optimize with intention theantiviral payload, vector, or cellular vehicle and/or dose to im-prove the chances of success. In light of this, investigators mustcontinue to test novel approaches to explore the potential mech-anisms. Possible in vivo mechanisms of action (MOAs) thatmay lead to clinical success include (1) selective outgrowth ofHIV-resistant cells to such a point that overall HIV replicationis thwarted (Lund et al., 1997; von Laer et al., 2006), (2) gen-eration of an expanding HIV-resistant T cell population throughspread of conditionally replicating HIV vectors (Weinberger etal., 2003), or (3) protection and/or boosting of critical HIV-spe-

cific immunity by HIV-resistant helper cells. A combination ofthese approaches may be required for success. Considering theMOA provides insight into why careful thought should go intothe design of the vector, or the site of action of the payload,depending on the hypothesized MOA of the therapy.

To explore the first MOA, one could accelerate the outgrowthof HIV-resistant cells in vivo. This can be done in several ways;the immediately obvious approach is to increase engraftment ofthe cellular dose either via repeat doses (T cells) or by opti-mizing conditioning before stem cell engraftment. Another wayto test this MOA when using T cells as the vehicle would beto create an environment for homeostatic expansion of modi-fied cells by “creating room” in the body by pretreatment con-ditioning (using antibodies or chemotherapy), or through thestrategic timing of leukapheresis and infusion. Alternatively, invivo selection could be performed for transduced cells ex-pressing the anti-HIV sequences. Methylguanine methyltrans-ferase (MGMT) has been used for selection of transduced HPCswhen used in combination with bischloroethyl nitrosourea(BCNU) and benzylguanine (BG) (Davis et al., 1997). WhereasBCNU has shown toxicity, the more recently available temo-zolimide, an oral agent that has been used clinically for pro-longed periods, has shown no serious adverse events. There-fore, in vivo selection could be a viable approach for improvingthe percentage of HIV-resistant cells in the body (Neff et al.,2003; Davis et al., 2004).

With the development of HIV-based lentiviral vectors (Nal-dini et al., 1996), it may be advantageous for these vectors tomobilize and spread their anti-HIV sequences throughout the Tcell population in the body (Dropulic et al., 1996; Bukovsky etal., 1999). The now completed VIRxSYS (Gaithersburg,MD)–University of Pennsylvania (Philadelphia, PA) clinicaltrial that used a conditionally replicating HIV-1-derived lentivi-ral vector expressing long antisense against the HIV envelopeprovides some intriguing data relating to this MOA. Interest-ingly, mobilization of the lentiviral vector via packaging by theendogenous HIV was detected in the serum of these patientsshortly after dosing, in the pattern of a short burst without sub-sequent detection of mobilized vector at later time points. Amodel describing the potential for conditionally replicating anti-HIV vectors to overcome wild-type infection in vivo has beendescribed (Weinberger et al., 2003). Further investigations intwo follow-on trials are ongoing in an effort to evaluate whetherthe mobilization is related to an antiviral effect, and whether itoccurs in lymphoid tissues such as the gut, which is a primarysite of HIV production (Veazey et al., 1998).

The safety of retroviral and lentiviral vectors for HIV/AIDShas been satisfactory to date and establishment of a crediblesafety database will be necessary for incorporation of these vec-tors into the practice of medicine. Although insertional onco-genesis was observed in one trial using retroviral vectors, thisevent is thought to have required the colluding circumstancesof a signaling payload and the cellular target for the leukemiato develop. On the basis of the adverse results with CD34� mar-row cells in severe combined immunodeficiency (SCID), it islikely that therapy involving HPCs may be more susceptible tothe risks of insertional mutagenesis than therapy involving ma-ture T cells. The extensive safety record of gene therapy withT cells, along with the demonstration of long-lived “centralmemory” and “stem cell” T cells, has increased the rationale

DROPULIC AND JUNE584

for T cell-based approaches (Fearon et al., 2001; Luckey et al.,2006). Thus, research will likely switch to lentiviral vectors inthe future as the supply of clinical-grade vectors improves. Es-tablishment of the safety of lentiviral vectors is in its infancy,but preclinical data demonstrating the lack of enhancer activ-ity and an altered insertional profile suggest that these vectorsare likely to be safer than retroviral vectors.

Gene therapy for HIV continues to march forward withpromise, and is sparking the evaluation of novel payloads anddosing approaches that can be applied in various areas of trans-lational medicine in gene therapy. Progress for the developmentof a gene therapy for HIV/AIDS has been slow, but it ispresently the only approach that promises a lasting cure for thedisease.

ACKNOWLEDGMENT

The authors thank Dr. Gwendolyn Binder for assistance inpreparing the manuscript for publication.

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Address reprint requests to:Dr. Boro Dropulic

Lentigen1450 South Rolling Road

Baltimore, MD 21227

E-mail: boro.dropulic@Lentigen.com

or

Dr. Carl JuneAbramson Family Cancer Research Institute

University of Pennsylvania421 Curie Boulevard

Philadelphia, PA 19104

E-mail: cjune@mail.med.upenn.edu

Received for publication March 20, 2006; accepted after revi-sion April 19, 2006.

Published online: May 22, 2006.

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